2.1. Introduction
The complex interaction between diet, normal intestinal microbiota, and wellbeing has encouraged the development of strategies to promote the selective proliferation of beneficial microorganisms into the gastrointestinal track of humans. Probiotics are microorganisms that positively affect human health with attributed powerful antipathogenic and anti-inflammatory properties (27) (Table 1).
TABLE 1Health Benefits of ProbioticsIntestinalImmunityReduce disease riskHelicobacter pyloriReducing allergicCoronary heart diseaseinfectionreactionsHigh blood pressureLactose intoleranceReducing opportunityUpper respiratoryIrritable bowel syndromeof infection bytract infectionsUlcerative colitispathogensUrinary tract diseaseCrohn'sdiseaseReduced cholesterolDiarrheaand lipidsConstipationAid in prevention ofStimulate mineralcolon canceradsorption
Also, years of probiotic research indicate that a selective modification of the intestinal microbiota and its associated biochemical activities can be promoted by selective prebiotics. Osborn D A, Sinn J K. Prebiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database of Systematic Reviews 2007. Prebiotics are non-digestible oligosaccharides (NDOs) that have a dual ability. First they reduce the intestinal colonizing efficiency of harmful bacteria and second they act as selective substrate to promote the growth and thereby increasing the number of specific probiotic bacteria.
In addition, an increasing number of studies have shown that probiotics work best when combined with prebiotics. Mayer et al. 2003 Research for creation of functional foods with Bifidobacteria. Acta Alimentaria 32 27-39. This combined form of delivery is known as a synbiotic. Gibson G R, Roberfroid M B. 1995 Dietary Modulation of the Human Colonic Microbiota—Introducing the Concept of Prebiotics. Journal of Nutrition 125:1401-12.
Galacto-oligosaccharides (GOS) are considered one of the preferred choices of prebiotics and in the gastrointestinal tract, GOS are resistant to enzymes and transit though the small intestine without being digested, but in the large intestine GOS are fermented and can activate growth of intestinal bifidobacteria such as Lactobacillus acidophilus and L. casei, hence acting as a prebiotic (26, 27, 37).
GOS are non-digestible oligosaccharides owing to the conformation of their anomeric C atom (C1 or C2), which allows their glycosidic bonds to evade hydrolysis by digestive enzymes in the stomach or small intestine. Free oligosaccharides are found in the milk of all placental mammals, providing a natural example of prebiotic feeding during infancy. According to the latest definition by the International Scientific Association for Probiotics and Prebiotics (ISAPP) “a dietary prebiotic is a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health” (30). The composition of human milk oligosaccharides (HMO) is very complex, which makes it unlikely to find alternative sources containing oligosaccharides of analogous composition. Improved colonic health among breastfed infants has been attributed to the presence of GOS in the mother's milk (2). In fact, infant formula with added GOS replicated the bifidogenic effect of the human milk with respect to metabolic activity of colonic microbiota and bacterial numbers (6, 21). Among non-milk oligosaccharides, GOS are of special interest as their structure resembles the core molecules of HMOs (3). However, GOS concentration and composition vary with the method and the enzyme utilized for their generation, which in turn may influence their prebiotic effects and the proliferation of colonic probiotic strains (29). Traditionally, GOS have been produced using β-galactosidases from mesophilic microorganisms. Mesophilic β-galactosidases require high initial concentrations of lactose to drive the reaction away from lactose hydrolysis and towards GOS synthesis. Since lactose is more soluble at elevated temperatures, thermostable β-galactosidases exhibiting high initial velocities and increased half-lives have been utilized to reach a favorable equilibrium for the transgalactosylation reaction (27, 37). However, competitive inhibition by glucose and/or galactose is another obstacle that remains which may be overcome by incorporating cells in the reaction (16, 20, 25, 27, 35).
The basidiomycete yeast Sporobolomyces singularis (formerly Bullera singularis) cannot utilize galactose to grow but proliferates on lactose due to the activity of its β-hexosyl-transferase (BHT, EC 3.2.1.21). Studies have shown that the BHT has transgalactosylation activity even at low lactose concentrations and very limited lactose hydrolysis. In addition, the enzyme does not appear to be inhibited by lactose concentrations above 20% and has the potential for conversions into GOS close the maximum theoretical of 75% (1, 9, 10, 28). Unlike β-galactosidases, the BHT from S. singularis simultaneously carries out glycosyl-hydrolase and β-hexosyl-transferase activities, converting lactose to GOS without extracellular accumulation of galactose. Two molecules of lactose are required during the transgalactosylation event: one molecule is hydrolyzed and the second acts as galactose acceptor, generating the trisaccharide galactosyl-lactose (β-D-Gal(1-4)-β-D-Gal(1-4)-β-D-Glc) and residual glucose. Galactosyl-lactose can also act as acceptor of a new galactose to generate the tetrasaccharide galactosylgalactosyl-lactose (β-D-Gal(1-4)-β-D-Gal(1-4)-β-D-Gal(1-4)-β-D-Glc), and similarly for the tetrasaccharide and subsequent products. The tri, tetra, and penta saccharides accumulating in S. singularis have been collectively designated GOS (9, 10).
For practical interests, a recombinant secreted BHT could have several advantages over the native enzyme, including improved large scale production and purification. Currently, purification of active enzyme from S. singularis requires cell lysis followed by multiple chromatography steps (1, 4, 16). Previous attempts to express recombinant β-hexosyl-transferase in E. coli BL21 have resulted in high levels of production, but the enzyme was inactive and insoluble (16).